Colony-stimulating activity in urine and serum in a child with cyclic neutropenia]

Colony stimulating activity (CSA) was estimated in daily urine samples and weekly plasma specimens of a 4-year-old girl with familial cyclic neutropenia. Nadirs of neutrophils and peaks of monocytes were found in regular cycles of 28 days. A close correlation was observed between nadirs of neutrophils and peaks of blood monocytes and colony stimulating activity in the urine. The role of colony stimulating factor as regulatory factor in granulopoiesis in cyclic neutropenia is discussed.     

Inhibitor of granulopoiesis in human cyclic neutropenia.

Peripheral blood mononuclear cells of a patient with cyclic neutropenia release a high molecular weight substance (over 300,000) inhibiting normal CFU-GM cells to enter the S-phase of cell cycle. The inhibitor was released predominantly in the neutropenic phase of the disease, while in the period of normal granulocyte count the release was lower or undetectable. Also sensitivity of patient's bone marrow CFU-GM cells to similar high molecular weight inhibitor produced by ML-2 cell liner or to human placental ferritin varied within the disease cycle. CFU-GM in the normal granulocyte count period were sensitive to the inhibitors, but CFU-GM in the neutropenic phase were resistant.     

Clinical effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on various types of neutropenia including cyclic neutropenia

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) was investigated for its clinical efficacy in the treatment of various types of neutropenia (3 cases with idiopathic neutropenia of suspected drug induction, 5 cases with idiopathic neutropenia of other origin, and 2 cases with cyclic neutropenia). Treatment with glycosylated rhG-CSF produced in the Chinese Hamster Ovary cells at dose levels of 2–5g/kg/day caused rapid increases of neutrophil counts associated with an improvement of the infection. In cyclic neutropenia patients, marked reduction in the duration of the neutropenic period was observed with rhG-CSF administration started before the period. Intercurrent stomatitis, which occurred in 1 patient, was markedly milder as compared to a previous episode which occurred during an untreated neutropenic period.The treatment of rhG-CSF was well tolerated and no adverse events were observed, nor was there any detectable anti-rhG-CSF antibody in any patients studied; hence the clinical use of rhG-CSF is considered to be safe.These results suggest beneficial effects of rhG-CSF on the recovery of neutrophil counts in cyclic and other types of idiopathic neutropenias, as well as for the treatment of neutropenia-associated infection.     

Regulation of cyclic nucleotide phosphodiesterase activity in human lung fibroblasts.

Cyclic nucleotide phosphodiesterase activity (3', 5'-cyclic-nucleotide 5'-nucleotidohydrolase, 3.1.2.17) was studied in homogenates of WI-38 human lung fibroblasts using 0.1--200 microgram cyclic nucleotides. Activities were observed with low Km for cyclic AMP(2--5 micron) and low Km for cyclic GMP (1--2 micron) as well as with high Km values for cyclic AMP (100--125 micron) and cyclic GMP (75--100 micron). An increased low Km cyclic AMP phosphodiesterase activity was found upon exposure of intact fibroblasts to 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase activity in broken cell preparations, as well as to other agents which elevate cyclic AMP levels in these cells. The enhanced activity following exposure to 3-isobutyl-1-methylxanthine was selective for the low Km cyclic AMP phosphodiesterase since there was no change in activity of low Km cyclic GMP phosphodiesterase activity or in high Km phosphodiesterase activity with either nucleotide as substrate. The enhanced activity due to 3-isobutyl-1-methylxanthine appeared to involve de novo synthesis of a protein with short half-life (30 min), based on experiments involving cycloheximide and actinomycin D. This activity was also enhanced with increased cell density and by decreasing serum concentration. Studies of some biochemical properties and subcellular distribution of the enzyme indicated that the induced enzyme was similar to the non-induced (basal) low Km cyclic AMP phosphodiesterase.     

The effect of aldosterone on cyclic nucleotide phosphodiesterase activity in toad urinary bladder

Cyclic AMP phosphodiesterase activity was assayed in the 700 xg supernatant solution of homogenates of epithelial cells scraped from toad urinary bladders. The activity of the enzyme was lower in cells obtained from bladders incubated with aldosterone for 24 hours than in cells from paired tissue incubated without aldosterone. This difference may well account for the permissive effect of aldosterone on the physiologic and biochemical responses of the toad bladder to vasopressin.     

B lymphocyte development in rabbit: progenitor B cells and waning of B lymphopoiesis

In mammals that use gut-associated lymphoid tissues for expansion and somatic diversification of the B cell repertoire, B lymphopoiesis occurs early in ontogeny and does not appear to continue throughout life. In these species, including sheep, rabbit, and cattle, little is known about the pathway of B cell development and the time at which B lymphopoiesis wanes. We examined rabbit bone marrow by immunofluorescence with anti-CD79a and anti-mu and identified both proB and preB cells. The proB cells represent the vast majority of B-lineage cells in the bone marrow at birth and by incorporation of 5-bromo-2'-deoxyuridine, they appear to be a dynamic population. PreB cells reach maximum levels in the bone marrow at 3 wk of age, and B cells begin to accumulate at 7 wk of age. We cloned two VpreB and one lambda5 gene and demonstrated that they are expressed within B-lineage cells in bone marrow. VpreB and lambda5 coimmunoprecipitated with the mu-chain in lysates of 293T cells transfected with VpreB, lambda5, and mu, indicating that VpreB, lambda5, and mu-chains associate in a preB cell receptor-like complex. By 16 wk of age, essentially no proB or preB cells are found in bone marrow and by PCR amplification, B cell recombination excision circles were reduced 200-fold. By 18 mo of age, B cell recombination excision circles were reduced 500- to 1000-fold. We suggest that B cell development in the rabbit occurs primarily through the classical, or ordered, pathway and show that B lymphopoiesis is reduced over 99% by 16 wk of age.     

Reduced variance of bone-marrow transit time of granulopoiesis—a possible pathomechanism of human cyclic neutropenia

Abstract. Human cyclic neutropenia (CN) is a haematological disorder characterized by oscillations in the numbers of neutrophilic granulocytes and other blood cells with a stable period of approximately 21 days. In most cases the neutrophils oscillate well below normal values such that these patients are chronically neutropenic. A comprehensive concept of the origin of CN is proposed. It assumes an abnormally small variance of the transit time of bone marrow cells (compared to normal human granulopoiesis) for the origin of the characteristic cycles. Furthermore, a reduced responsiveness of the immature granulopoietic bone marrow cells to the mitotic feedback stimuli is assumed to account for the subnormal neutrophil peaks. Together with feedback control provided in a simulation model of normal human granulopoiesis these two abnormalities can explain experimental and clinical cell kinetic data for bone marrow and blood in CN.     

Catecholase activity associated with copper-S100B

This study addresses the spectroscopic properties and reactivity associated with the copper-loaded form of S100B isolated from bovine brain. Copper(II)-S100B displays EPR features typical of a type II copper center and is shown here to exhibit catecholase activity, the two-electron oxidation of catechols. The steady-state kinetics associated with the oxidation of several catecholamines has been probed in order to further characterize this activity. The evidence provided indicates that the catecholase chemistry is copper initiated. Superoxide dismutase has no effect on the rates of catecholamine oxidation catalyzed by Cu-S100B, establishing that superoxide is not produced during this reaction, ruling out an autoxidative mechanism. Addition of catalase to the Cu-S100B reaction with catechols reduces the amount of oxygen consumed by 50%, demonstrating that peroxide is released during this reaction. The release of peroxide is mechanistically distinct from the type III dinuclear copper proteins, catechol oxidase and tyrosinase.     

Regulation of RuBP carboxylase activity associated with photo-inhibition of wheat

Detached wheat leaves were illuminated in air until a steady rate of photosynthesis was established. Then the gas was changed to 1% O₂, 99% N₂ and after 2.5 h further illumination the capacity of the leaves for photosynthesis in air was decreased to approximately 50%. Measurement of RuBP carboxylase activity in extracts showed that inhibition of photosynthesis was accompanied by 70% inactivation of this enzyme. The capacity for photosynthesis and the activity of RuBP carboxylase were recovered when leaves were returned to normal air. Extracts of the leaves made when photosynthesis and carboxylase activity were low, recovered most of the lost carboxylase activity when supplemented with bicarbonate and magnesium ions. The time courses for activation and inactivation of the RuBP carboxylase in these experiments suggests the operation of a mechanism that has not yet been elucidated.     

Purification and characterization of proteins with associated tyrosine protein kinase activity from human B lymphocytes

Burkitt lymphoma cells and their counterpart of normal origin contain proteins with associated tyrosine protein, kinase activity. These proteins were isolated by affinity chromatography and Fast Pressure Liquid Chromatography. Proteins with enzyme activity had an app. M. W. of 47 KDa. This protein in extracts of Burkitt lymphoma cells differed by overall charge and phosphorylation from the 47 KDa protein isolated from B lymphocytes of normal origin. Before and after purification the 47 KDa protein of Burkitt lymphoma cells reacted with an antibody directed against the dodecapeptide Arg-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg (conserved region of pp60src), the 47 KDa protein from B cells of normal origin did not; the same protein from both cell lines reacted with anti-pp60src antibody. These results suggest that a tyrosine protein kinase, related to the products of the src family of oncogenes, is modified in Burkitt lymphoma cells.     

Regulation of estrogen activity in human endometrium: effect of IL-1beta on steroid sulfatase activity in human endometrial stromal cells

We investigated the effect of interleukin 1beta (IL-1beta) on steroid sulfatase (STS) activity and the expression of STS mRNA in human endometrial stromal cells. Endometrial tissue samples were obtained from patients undergoing hysterectomy to remove uterine fibroids. Stromal cells were isolated from the tissue preparation and cultured. IL-lbeta (1 approximately 100 ng/ml) was added into the culture medium and incubated for 24 h. The expression of STS mRNA was measured by competitive RT-PCR. The addition of IL-lbeta at 10 and 100 ng/ml suppressed STS mRNA expression to 55.2 +/- 12.8% and 25.1 +/- 10.9%, respectively, of the control sample to which no IL-lbeta had been added. STS activity was measured by radiolabelled steroid metabolite using thin layer chromatography, and this activity was also significantly suppressed in response to the administration of IL-lbeta in a dose-dependent manner. When IL-1 receptor antagonist (IL-1ra) was added together with IL-1beta to the culture medium, mRNA expression and STS activity were recovered. The present study is the first to demonstrate IL-1beta regulation of STS activity locally in human endometrium. IL-1beta suppressed mRNA and activity of STS in stromal cell culture. This initial demonstration of IL-1beta regulation of STS implies that IL-1beta may control the steroid microenvironment in human uterine endometrium by reducing biologic action of estrogen.     

Regulation of human in vitro anti-hapten responses: demonstration of a carrier effect

Human peripheral blood mononuclear cells, tonsillar lymphocytes, or mixtures of allogeneic or autologous B and T cells from these tissues were stimulated in vitro with the soluble hapten:carrier complexes TNP-OA, TNP-KLH, TNP-Myo, or TNP-Lac. These complexes were able to induce TNP-specific, direct PFC during 5–6 days in culture. The response involved proliferating PFC precursor B cells, nonproliferating T helper cells, and radiosensitive T-suppressor cell precursors. Exposure to high concentrations of free or carrier-bound hapten resulted in the inactivation of PFC precursor B cells. Carrier specific suppressor T cells could be induced by priming with nonhaptenated carrier protein and were able to block the PFC response when added to fresh target cultures. The use of hapten:carrier complexes permits the characterization of distinct T-cell functions independently of the assay system for B-cell activation.     

Metallocarboxypeptidases: a cadmium-carboxypeptidase B with peptidase activity

The replacement of zinc by a series of other metal ions (Co, Cd, Mn) resulted in enzymatically active carboxypeptidases both as peptidases and esterases. The effect of the metal replacement on the kinetic parameters varies for the various substrates. Cd-CPB, previously known for its lack of peptidase activity, shows enhenced activity as long as the substrate interact with four subsites (including the N-terminal blocking group). Kinetic measurements and chemical modification revealed differences in the nature of the residues necessary for the proper alignment along the active site between the Zn and Cd carboxypeptidases. A simple explanation of the data observed is based on the assumption that replacement of the metal affects the microenvironment of the active site.